Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function

Paula Schiapparelli, Natasha L. Pirman, Kyle Mohler, Pierre A. Miranda-Herrera, Natanael Zarco, Onur Kilic, Chad Miller, Sagar R. Shah, Svetlana Rogulina, William Hungerford, Laura Abriola, Denton Hoyer, Benjamin E. Turk, Hugo Guerrero-Cázares, Farren J. Isaacs, Alfredo Quiñones-Hinojosa, Andre Levchenko, Jesse Rinehart

Research output: Contribution to journalArticlepeer-review


Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.

Original languageEnglish (US)
Article number109416
JournalCell reports
Issue number3
StatePublished - Jul 20 2021


  • SPAK
  • WNK1
  • glioblastoma cell migration
  • kinase
  • small-molecule kinase inhibitor
  • synthetic biology

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


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